[CANCER RiiSI'ARCM 53. 427.V42H<>.September 15. ITO]

Antitumor Protection from the Murine T-Cell Leukemia/Lymphoma EL4 by the

Continuous Subcutaneous Coadministration of RecombinantMacrophage-Colony Stimulating Factor and Interleukin-21

Daniel A. Vallera,2 Patricia A. Taylor, S. Lea Aukerman, and Bruce R. Blazar

Department of Therapeutic Radiology. Section on Kxperitnenttil Cancer Inwiunolo^v ¡D.A. V.¡,muíDepartment of Pediatrics, Division of Hone Marrow Transplantation ¡P.A. T.,H. R. lì.j.University of Minnesota Hosfñtiilami Clinic, Minncaptdi.s, Minnesoui 5.V55. and Chiron Cttrporalion, i'.mcr\\:ilU-, California V-JMW/S, L. A.¡

ABSTRACT

Combined continuous s.c. Coadministration of macrophage-colonystimulating factor (M-CSF) plus interleukin-2 (IL-2) by osmotic pumpprotected mice given i.v. injections of a lethal dose ulT.1.4 T-cell leukemia/

lymphoma. Antitumor protection was significantly greater than that afforded by treatment with either cytokine alone. Since neither IL-2 receptors nor M-CSF receptors were expressed on EIA the antitumor effectwas likely attributed to murine effector cells. To determine how M-CSF +IL-2 provided this effect, we performed immunophenotypic and func

tional analyses as well as in vivo depletion studies of putative antitumoreffector cells. Splenic phenntyping experiments revealed that the highestlevels of macrophages and natural killer cells were observed in mice giventhe cytokine combination rather than either M-CSF or IL-2 alone. In vivo

depletion of natural killer cells ablated the antitumor protective effect ofM-CSF and IL-2. T-cells were also important for M-CSF + IL-2 efficacy,since adult thymcctomy/T-cell depletion significantly inhibited the ability

of cytokine Coadministration to protect against EL4. Coadministration ofthe 2 cytokines significantly elevated in vivo levels of CD3*CD4*,CD3*CD8*, CD.VNK1.1* T-cells, and CD3MTD25* (activated) T-cells, and

elevated anti-1 I 4 cytotoxic T-cell activity measured in vitro. AlthoughWBC counts and fluorescence-activated cell sorter studies showed thatM-CSF t IL-2 treatment significantly elevated neutrophils, s.c. delivery ofgranulocyte-colony stimulating factor at doses sufficient to induce neutro-philia was unable to confer anti-EL4 protection. These studies indicatethat macrophages, T-cells, and natural killer cells are all important in theM-CSF + IL-2 ¡iiiii-l'.l.4 response. The superior antitumor effect of this

cytokine combination along with the ability of M-CSF to diminish thetoxicity of IL-2 in this model suggests that further investigations into the

clinical potential of this combination treatment are warranted.

INTRODUCTION

Macrophagc-colony stimulating factor is a glycoprotcin that acts on

progenitor and mature cells of monocytoid lineage via specific cellsurface receptors (1). M-CSF1 receptor (c-fms) is distributed mostly

on monocytcs/macrophages, myeloid precursors, and placenta! tro-phohlasts. Thus, M-CSF stimulates murine macrophages to secrete avariety of products important in the immune response (2-4). M-CSF

induces enhanced expression of Fc receptors (5), cytotoxic activity ofmacrophages and monocytes (6), enhanced chemotaxis (7), and respiratory hurst activity (8), and can he used for viral immunotherapy(9). In murine models, M-CSF has resulted in tumoricidal activity

Received 3/26/«3;accepled 7/8/93.The costs of publication of this article were defrayed in part hy the payment of page

charges. This article must therefore he hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part hy USPHS Grants PO1-CA-21737. ROI-CA-31618.RO1-CA-36725, and NOI-A1-85M2, awarded by the National Cancer Institute, Department of Health and Human Services. Center for Experimental Transplantation and C'ancer

Research Publication 75. B. R. B. is a recipient of the Edward Mallinckrodt. Jr.. Foundation Scholar Award.

2 To whom requests for reprints should be addressed, at Box 367 UMHC, 420 Dela

ware Street S.E.. Minneapolis. MN 55455.3 The abbreviations used are: M-CSF. maerophage-colony stimulating factor; Ci-CSF,

granulocyte-colony stimulating factor; IL. intcrlcukin; I.AK. lymphokine activated killer;ATCC. American Type Culture Collection; CTL. cytotoxic T-lvmphocvtc; IF. immuno-fluorescence; PBS, phosphate-buffered saline; FACS. fluorescence-activated cell sorter;

E:T, effectortarget; FITC. fluorescein isothiocyanate; MST. mean survival time; NK.natural killer.

when used alone (10) or in combination with other therapies (11).Monocytes/macrophages are important in antitumor responses. In theafferent phase of response, macrophages process antigens and presenttumor-specific peptides in conjunction with major histocompatibilitycomplex antigens to the T-lymphocyte. Macrophages can also regulate

lymphocytes by releasing stimulatory and inhibitory cytokines. In theefferent phase, macrophages can serve as important effector cells inantitumor responses.

IL-2 receptors are distributed on T-cells, B-cells, monocytes, myeloid precursors, and NK cells. Cells that are targets for IL-2 have also

been known to play a very important role in antitumor immune responses. LAK cells with IL-2 treatment have been used for cancertherapy (12-15). Circulating NK cells have been proposed as thecirculating in vivo cytotoxic effectors in studies in which IL-2 hasbeen administered for cancer therapy (16-20).

In these studies, we tested the ability of Coadministration of M-CSF+ IL-2 to heighten antitumor responses. Following i.v. injection of theT-cell leukemia/lymphoma EL4, we observed that there is an enhancement of antitumor protection with M-CSF + IL-2 that is significantly

greater than that observed with either cytokine alone. Then, usingimmunophenotyping, in vivo depletion, and other immunologicaltechniques, we also evaluated the effector cells responsible for theantitumor effect.

MATERIALS AND METHODS

Mice and Tumors. Female 5-8-week-old C57B1/6 mice (Thyl.21). orB6.PL-i/iv-/VCy mice (Thy 1.1 ' ) were obtained from The Jackson Laboratory

(Bar Harbor. ME) and used for all experiments. The chemically inducedC57BI/6 EL4 lymphoma (21) originally obtained from the ATCC (Rockville.MD) was propagated in suspension culture in RPMI 1640 supplemented with10% heat-inactivated fetal call serum, 2 HIM glutamine. and 1(K) U/mlpenicillin/streptomycin at 37°Cin a 5% COi/y5% air atmosphere. We have

designated the subline used in these studies as EL4/University of Minnesota.Antibodies for IF Studies. Antibodies tor IF studies included: anti-Thy 1.2

monoclonal antibody (clone 30-HI2. rat anti-mouse lgG2b from ATCC) (22),anti Ly-1, (clone 53-7.313, rat IgG2a from ATCC) (22), anti-Ly2.2 (clone 2.43,

rat lgG2b from Dr. Frank Fitch, University of Chicago, Chicago, IL) (23),anli-CD4 (clone OKI.5 also from Dr. Fitch) (24), anti-CD2 (clone RM2.2, ratIgG from Dr Vagita, Juntendo University. Tokyo. Japan) (25), anti-CD3-t

(clone 1452C11. hamster IgG from Dr. Jeifcry Bluestone. University of Chicago, Chicago, IL) (26), anti-B22() (clone RA3-6B2, rat IgG2a from ATCC)(27). anti-ncutrophil (clone RB6-8C5. rat IgG2b from Pharmingen) (28), anti-Lyb8.2 (clone CY-34-1.1, mouse IgGl, from Dr. Kevin Holmes. N1H) (29),anti-LFA-1 (clone FD441.8, rat lgG2b from Dr. Frank Fitch) (30), anti-macrophage/monocyte (clone F4/8I), rat lgG2b provided hy ATCC) (31). anti-IL-2 receptor (clone 7D4, rat IgG from Pharmingen) (32), and NK1.1 (clone

PK136 mouse IgG2a from Dr. Gloria Koo, Rahway, NJ) (33). The monoclonalantibodies were linked to FITC or to phycoerythrin for 2-color IF as reported

(34). Direct IF studies were performed as previously described, hy incubatingEL4 cells for l h at 4°Cwith an optimal concentration of labeled antibody.

Cells were washed 3 times and then suspended in (1.5 ml of lrr paruformal-

dehyde. Binding was quantitated on a FACScan (Becton Dickinson. MountainView, ÇA).A minimum of 2(),(KM)cells (determined by forward light scatter)was analyzed. Anti-human CD7 (3Ale, a mouse IgG2b provided by Dr. BartonHaynes. Duke University, Durham, NC) was conjugated to FITC or phyco-

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ANTITUMOR EFFECTS WITH M-CSF + II.-2 THERAPY

erythrin and used to determine the degree of background binding. Backgroundbinding was subtracted and the percent of specific binding was calculated asdescribed (34).

Tumor Localization in Congenie Mice. Groups of the B6.PL-i/iy-/A/Cycongenie mice (n = 4) were given i.v. injections of 2 X IO6 Thy 1.2+ EL4

cells. The C57BI/6 congenie strain expresses the Thy 1.1 surface markerinstead of Thy 1.2. Thus, injected EL4 cells can be detected by IF. Injected micewere sacrificed on days 7 and 14 posttumor inoculation. Bone marrow, lymphnodes, and thymocytes were passed through wire mesh, washed twice, andsuspended. Peripheral blood was collected in heparin, enriched with a Ficollgradient, washed, and then suspended. RBC in the spleen preparation werelysed. Splenocytes were then washed and suspended. Cells were reacted withanti-Thyl.2-FITC and control 3Ale-FITC. Direct IF was determined on the

FACScan as described.Cytokines. M-CSF is a highly purified recombinant human protein pro

duced in Excherichia coli (35) by Cetus Corp. (Emeryville. CA). The E.ro//-produced recombinant M-CSF is nonglycosylated. The specific activity is1 X IO7 units/mg. Rccombinant IL-2 produced in E. coli was provided by Dr.

Maurice Gately (Hoffman-LaRoche, Nutley, NJ). Human IL-2 is nonglycosylated with a specific activity of 1.2—1.7X If)7 units/mg. Human G-CSF is a

highly purified recombinant human protein produced in yeast by the ImmuncxCorp (Seattle, WA).

In Vivo Administration of Recombinant M-CSF and Recombinant IL-2.For continuous s.c. delivery of cytokines. 14-day miniosmotic pumps (ALZA

Corp., Palo Alto, CA) were implanted under general anesthesia as described(36) in control and experimental mice, on day -3 (with day 0 the day of i.v.tumor inoculation); HP EIA cells were given. Two hundred-jj.1 volumes M-CSF and recombinant IL-2 were injected into the pumps. Control pumps were

injected with PBS. The pumps were implanted in the dorsal lumbar area.Hematological Evaluation of Recipients. Fifty //I of peripheral blood

were obtained by retro-orbital venipuncture on days 7, 14, and 28. Leukocytenumber and morphology were determined by cxaminination of Wright-Giemsa

stained slides (37).Lysis of Targets by CTL, NK Cells, and LAK Cells. Mice were given

implants of pumps containing M-CSF, IL-2, M-CSF + IL-2, or control PBS,

as described. Groups of mice were given injections of EL4 tumor cells andsacrificed on day 7 postinjection, at which time splenocytes were isolated andtested for their ability to lyse MCr-labeled target cells. Splenocytes were pooledfrom 3 mice. Cytolysis of H-2h EL4 cells (by specific CTL), of H-2" YAC-1cells (by NK cells), and of H-2d P815 cells (by LAK cells) was measured at

effectortargcl ratios of 50:1, 25:1, 12.5:1, and 6.25:1. Percent cytotoxicity wasdetermined as described previously (38). For EL4, spontaneous and maximumrelease was 470 and 4957 cpm, respectively. For YAC-1 targets, spontaneous

and maximum release was 551 and 5443 cpm, respectively. For P815 targets,spontaneous and maximum release was 327 and 3576 cpm, respectively.

Statistical Analyses. Groupwisc comparisons of continuous data weremade by Student's I test. For plotting actuarial survival, the computer program

for compiling life table and statistical data analyzed by a log-rank test was

written by Dr. Bruce Bostrom, Department of Pediatrics, University of Minnesota.

RESULTS

Flow Cytometric Analysis of EL4 Reveals Absence of IL-2 andM-CSF Receptor. EL4 cells were reacted with a panel of labeledmonoclonal antibodies directed against lymphoid cell surface antigens. EL4 cells expressed the following markers: 95% Thy 1.2, 96%Lyl, 85% CD2, 93% CD3, and 96% CDlla (LFA-1) surface markers,which are all associated with T-cells. The cells did not express B-cellantigens or the low affinity IL-2 receptor (CD25). As a control, CD25was found to be present on 88% of day 3 phytohemagglutinin-acti-vated splenocytes, but only 7% of nonactivated splenocytes. IL-2(1000 units/ml) or M-CSF (2000 units/ml) was unable to stimulate

EL4 cells. No proliferation as measured by incorporation of tritiatedthymidine was observed on day 1 or 2.

Determination of a Tumor Dose. To determine the susceptibilityof the C57B1/6 mice to EL4 tumor, groups of mice were given injections of varying doses of EL4 tumor cells (n = 10/group). Survivalcurves generated over 100 days (post-tumor injection) showed that a

dosage of 10s cells/mouse was mostly lethal. After 100 days, IO4

cells/mouse was an 83% lethal dose (a dose at which 83% of the micedied), 1(P cells/mouse was a 63% lethal dose, and IO2 cells/mouse

was a 15% lethal dose. There was little, if any, difference in the doseresponse curves of mice given injections by either the i.v. or i.p. route.

Tumor Localization Studies. To determine the localization ofThy 1.2* EL4 tumor cells, tumor was injected into Thy 1.1 + congeniemice. On day 7 after i.v. injection of 2 X IO6 tumor cells, no tumor

was detected in bone marrow, lymph node, peripheral blood, spleen,or thymus, suggesting that the levels of propagation were not sufficient for detectability. On day 14, tumor appeared in bone marrow,lymph node, and/or peripheral blood, but not the spleen or thymus in3 of 4 mice. On day 21, tumor was in the spleen and thymus of 2 of4 mice. No reactivity was observed when anti-Thyl.2-FITC was re

acted with bone marrow, lymph node, peripheral blood, spleen, orthymus from B6.PL-//iy-7A/Cy mice. Evaluation of preterminal

C57B1/6 mice receiving i.v. injections of EL4 in other experiments hasfrequently demonstrated the presence, at autopsy, of EL4 tumor in theperitoneum, mesentary, uterus, kidney, liver, and muscle.

Low Dose IL-2 Administration Elicits Ant ¡tumorEffect. To testthe ability of s.c. administered IL-2 to protect mice against EL4lethality, IL-2 was administered to mice given IO5 EL4 cells (Fig. 1).

Pumps were inserted on day -3 so that circulating IL-2 levels would

presumably reach an equilibrium state at the time of EL4 injection onday 0. Mice given either 0.625 /J-g/day or 2.5 fig/day in 14-day pumps

showed significant (P < 0.02), albeit partial, EL4 protection compared to PBS-treated controls. In both groups, 40-50% of the mice

succumbed to the EL4 tumor by day 60 postinjection. In a secondexperiment (n = 15/group) shown in Fig. 2A, higher dosages of IL-2(5 jug/day) in 14-day pumps were studied. The higher dosage resulted

in significant toxicity, with the majority of the animals dead by day 10postinjection. The mean survival time of these mice was significantly(P < 0.009) shorter (14 days) than control PBS-treated mice.

Coadministration of M-CSF and IL-2 Protects against Tumorand Reduces IL-2-mediated Toxicity. To test our hypothesis thatmonocytes, NK cells, and T-cells together augment antitumor effectors, we coadministered M-CSF and this higher dose of IL-2. Survivalwas significantly (P < 0.0005) improved in comparison to PBS-treated controls (Fig. 2A). Seventy % of the M-CSF + IL-2-treated

mice survived to day 100 (MST > 100 days). Some antitumor protection was conferred in this experiment by 20 pig/day M-CSF treat-

p<0.02 compared to PBS

mco

Iso.

60 70 80 90

Days

Fig. 1. Continuous s.c. administration of low dose IL-2 by pump partially protectsagainst EL4 lumorigenicity. Groups of IO C57BI/6 mice were given pumps containingIL-2 on day -3 and injections of IO5 EL4 cells i.v. on day 0. Actuarial survival was plotted.

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ANTITUMOR EFFECTS WITH M-CSF + IL-2 THERAPY

Fig. 2. Cnnlinuous s.c. aüminislralionof M-CSF+ IL-2 by pump protects against F.t.4 tumorigenic-ily. Groups of C57B1/6 mice were given pumpscontaining M-CSF + IL-2, M-CSF, IL-2, or PBSon day -3 and injections of IO5 EL4 cells i.V. onday 0. Independent experiments (n = 15/group)are shown in A and R and the pooled data (n =

30/group) arc shown in C. Actuarial survival wasplotted.

CO

oCLO

A. Experiment 1

*p<0.05 compared to PBS or IL-2

•¿�M-CSF+IL-2•¿�

1.0V0.9-

0.8-

0.7-

0.6-

0.5-

0.4-

0.3-

0.2-

0.1-

0.0

10 20 30 40 50 60 70 80 90 100

B. Experiment 2C g tf *p<0.003 compared to PBS

m £ a 'M-CSF+IL-2

M-CSF

PBS

n=15/group •¿�IL-2

10 20 30 40 50 60 70 80 90 100

C. Pooled Data*p<0.03 compared to PBS

10 20 30 40 50 60 70 80 90 100

•¿�M-CSF

ment alone (40% survival at day 100, MST = 46 days), which was

significantly (P < 0.003) higher than protection obtained in the control PBS-treated group (MST = 34 days).

In a different experiment (Fig. 2B), groups (n = 15) of mice treated

identically to those in Fig. 2A showed similar results. Early deaths dueto toxicity were observed with IL-2 treatment as compared to PBScontrols. The mean survival time of the IL-2-treated mice was 32 daysshorter than that of the PBS-treated mice. M-CSF + IL-2 treatment

gave the best antitumor protection with nearly 90% of the mice alive

more than 90 days after tumor injection. The level of protection wassignificantly greater than that obtained with M-CSF alone (P =0.0026), IL-2 alone (P = 0.000005), or PBS (P = 0.(K)27) treatment.In contrast to the experiment in Fig. 2A, M-CSF treatment alone did

not protect in this experiment. Here, not all of the animals succumbedto EL4 in the PBS control-treated group.

Since the experiments in Fig. 2, A and B, were performed identically, the data were pooled in Fig. 2C (n = 30/group). M-CSF +IL-2-treated mice showed 78% survival 100 days (MST > 100

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ANTITUMOR EFFECTS WITH M-CSF + IL-2 THERAPY

Table I Coadminisiraiion .v.r. of M-CSF + IL-2 enhances neutrophil in the peripheralblood of EL4-injecled mice

C57B1/6 mice were given 10s EL4 cells i.v. on day 0. On day -3, 14-day pumps

containing 20 ¿tg/dayM-CSF + 5 fig/day IL-2 were implanted. On day 11, mice werehied by rctro-orbiial ve nipu nelure. Smears were prepared, stained, and read. Mean values±1 SD are reported x HT3/^!.

CellsFig.

2ßexperimentWBCANC'ALCnFig.

2AexperimentWBC'ANCALCnPBS13.6

±5.13.4±1.711.2

±2.8158.9

±2.10.8±0.38.0±1.815M-CSF17.6

±3.7"5.3±3.1°12.3

±3.41510.4

±3.31.9±0.9"8.4

±3.015IL-210.15.25.0114.2

±4.3°5.1±2.7"8.0

±1.95M-CSF

+IL-237.4

±35.3°-''21.0±25.1"'''16.5

±11.61515.5

±11.2"6.4±l.ff-b8.1

±5.214

•¿�P < 0.04 as compared to PBS controls.* P < 0.05 as compared to M-CSF group.' ANC, absolute neutrophil counts; ALC, absolute lymphocyte counts.

days) post-tumor injection, and survival was significantly (P <0.0000005) better than the IL-2 alone-treated group (MST = 10days), the M-CSF-treated group (P < 0.003) (MST = 46 days), orthe PBS-treated control group (P < 0.00001) (MST = 36 days). TheIL-2-treated mice showed significantly worse (P < 0.009) survivalthan the PBS-treated controls. Mice given M-CSF treatment alone

showed significantly better survival (P < 0.03) than PBS controls,but mean survivals differed by 10 days in the 2 pooled groups. Together, these experiments show that M-CSF + IL-2 coadministrationis effective in conveying antitumor protection and that M-CSF insome way protects against the toxicity conferred by the higher IL-2

dose treatment.Coadministration of M-CSF and IL-2 Stimulates Peripheral

Blood Neutrophilia. To determine the effects of M-CSF + IL-2

treatment on peripheral blood cell populations, the mice in Fig. 2, Aand B, were evaluated at the end of infusion (day 11) for WBC counts,absolute neutrophil counts, and absolute lymphocyte counts (Table 1)(day 11 is the last day of infusion, since 14-day pumps were insertedon day -3.) There were enough mice available from the PBS-, M-

CSF-, and M-CSF + IL-2-treated groups for hematological analysison day 11. However, there was only one survivor from the IL-2-treated group in Fig. 2fi, and 5 mice from the IL-2-treated mice in Fig.2A. In both experiments, M-CSF + IL-2 treatment significantly (P <0.04) elevated WBC levels in comparison to PBS-trcatcd controls, andthe M-CSF- and IL-2-treated groups showed higher WBC values thanthe IL-2 or M-CSF treatment alone. In fact, in the mice from Fig. 2B,mean WBCs were elevated about 3-fold in the M-CSF + IL-2-treated

group as compared to the PBS control. In both experiments, the WBCelevation in the M-CSF + IL-2-treated group was the result of a

significant (P < 0.04) increase in peripheral blood neutrophil. but notlymphocyte, levels.

Elevated neutrophil levels induced by M-CSF + IL-2 were notresponsible for the protective anti-EL4 effect in vivo, since G-CSF, a

powerful enhancer of neutrophil number and function, was not able toprotect mice given G-CSF by pump. In the experiment shown in Fig.2/4, an additional group of mice given G-CSF died at a similar rate tomice given PBS (Fig. 3). The P value (P = 0.34) derived from the

comparison of the 2 groups was not significant. The neutrophil levelsmeasured on day 11 (also shown in Fig. 3) in the G-CSF mice were

significantly higher than in the PBS mice. In fact, higher levels ofneutrophil stimulation were achieved in the G-CSF mice than in themice given M-CSF + IL-2 which were subsequently protected from

the EL4 tumor.M-CSF and IL-2 Coadministration Stimulates Splenic Local

ized NK Cell and Macrophage Compartments in Normal andTumor-injected Mice. To further explore the cell populations affected by M-CSF + IL-2 treatment, normal animals that had received

pumps were sacrificed on day 7 and the spleen cells were isolated andexamined by FACS (Table 2). These studies revealed about a 2.0- to2.5-fold increase in T-cell populations (CD3 +, CD4+, CDS+ ) in mice

given M-CSF + IL-2 as compared to the PBS control. A similarelevation was noted with IL-2 administration alone. In comparison toIL-2 treatment alone, M-CSF + IL-2 treatment elevated the cell

numbers in the macrophage compartment ( 181 X IOV/J.1versus 116 X10-V/J.l)and in the NK compartment (106 X 103/jLtIversus 79 X

lOV/nl). Similar observations were made on day 7 with EL4-injected

Fig. 3. Continuous s.c. administration of G-CSFby pump does not protect against EL4 tumori-

genicily. In addition to the groups shown in Fig. 14,a group of mice was given 14 day pumps containing I fig/day G-CSF on day -3 and injections ofIO5 EL4 cells i.v. on day 0. Fourteen mice were

included in this group. Actuarial survival was plotted. Also, mice were bled on day 11. to determinethe ability of G-CSF to elevate neutrophils. WBCcounts and differentials were performed as in Table1. and the data arc shown.

IW

o

oQ.

I

1.0H0.9-n

oU.o0.7-0.6-0.5-0.4-0.3-0.2-0.1

-LIL-jitI*[1>-i1L••f¡*"frL*+PBS

&CSF M-CSF+IL-2WBC

13.6±5.1 61.4±13.3-37.4±35.3-ANC

3.4±1.7 47.0±10.5- 21.0125.1*ALC

11.2±2.8 14.1±4.816.5111.6n

15 1414•p<0.04•M-CSF+IL-2*

G-CSFPBS

10 20 30 40 50 60 70 80 90 100

Days4276

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ANTITUMOR EFFECTS WITH M-C'SF + IL-2 THERAPY

Table 2 Coadministration s.c. of M-CSF and IL-2 enhances various leukocyte suhpttpulations in the spleen of normal mice as measured by FACS

C57BI/6 mice were given 14-day pumps containing 20 ng/day M-CSF -I-5 /tg/day IL-2. On days 7 and 14. spleens were removed. Three mice/group were studied with the exceptionof the lL-2-lrcated group, in which there was only one survivor al day 7. Cells from each group were pooled. Samples of cells were treated with various fluorochrome-lahcled monoclonalantibodies for I- or 2-color immunofluorcscencc as described in "Materials and Methods" and percent positive cells were determined by FAC'S. Percentages were multiplied by thetotal spleen count and values are reported X 10"" cells/spleen. Since 14-day pumps were inserted on day -3, day 14 was chosen for study since cytokine delivery had ceased at this

time.

Granulocytc cell,8C54markerDay

7M-CSF+IL-2IL-2M-CSFPBSUfi

NopumpDay

14M-CSF-1-IL-2IL-2M-CSFPBSBfi

No pump354512135111332114Macrophage

cell,F480' markerISIUfihl58379533•175042N

K cell,NK1+ CDS"marker1067913i)4523232115B-cell,B220*marker1241165061493930613947T

cellsCD3+

NKP1151203146333724403838CD4+CD8+ CD3+ IL-2R+534914241817131921212737915109101314133541784117854CD3+NK1*4449573114643

animals (Table 3). On day 14, responses had normalized since this was3 days past the delivery lifespan of the pumps. The FACS studiesdemonstrate that the addition of M-CSF to IL-2 resulted in the stimu

lation of multiple lineages of cells with antitumor capabilities including macrophages, T-cells, and NK cells.

NK Cells Are Required for M-CSF + IL-2-mediated Protectionin EL4-injected Mice. To determine whether NK cells were responsible for the M-CSF + IL-2 effect, groups of mice (n = 10/group)were NK cell-depleted by administrating a known depletionary doseof 400 jj,g anti-NK 1.1 monoclonal antibody 4 days before tumor in

jection and then weekly thereafter for 2 additional injections (Fig. 4).A group of mice was depleted of T-cells by adult thymectomy followed by a depleting dose of anti-CD4 plus anti-CD8 monoclonal

antibody (400 /j,g each). When mice depleted in this fashion weregiven 10s EL4 cells i.V., the NK cell-depleted animals died earlier

(MST = 24 days) than control mice given PBS (MST = 31 days)

(P < 0.006). These earlier deaths were unaffected by the administration of M-CSF + IL-2. Since the nondepleted control mice givenM-CSF + IL-2 were protected against EL4-related mortality, these

data argue that NK cells were at least one of the antitumor effectorcells affected by M-CSF and IL-2 treatment. The removal of T-cellsalso significantly (P < 0.03) reduced M-CSF + IL-2 efficacy, indicating that T-cells are also involved ¡nM-CSF + IL-2-mediated pro

tection.M-CSF + IL-2 ( «.administration Augments NK and CTL

Splenic Lytic Activity in EL4-injected Mice. On day 7 after tumor

injection, splenocytes were removed and tested for their activityagainst NK 5lCr-labeled target cells (YACÕ)or against CTL 51Cr-

labeled targets (EL4 cells). The cytolytic response to YACÕtarget

cells was elevated in both M-CSF + IL-2-treated mice and IL-2-

treated mice, as compared to PBS controls. At an E:T ratio of 25:1,EL4-injected mice treated with M-CSF + IL-2, IL-2, M-CSF, or PBS

showed cytolytic values of 25, 17, 5, or 5%, respectively. At an E:Tratio of 12:1, EL4-injected mice treated with M-CSF + IL-2 or IL-2alone showed cytolytic values at least 6.5-fold and 4.7-fold greaterthan PBS controls, respectively. Again, M-CSF treatment alone did

not elevate NK response. Similar responses were observed in treatedmice not given EL4.

CTL response (measured with the labeled EL4 cells) was elevatedonly by M-CSF and IL-2 treatment. At an E:T ratio of 50:1, micegiven injections of H)5 EL4 cells and treated with M-CSF + IL-2,

IL-2, M-CSF, or PBS showed cytolytic values of 27, 6, 1, or 3%, respectively. Mice given injections of IO4 EL4 cells and treated with

M-CSF + IL-2, IL-2, M-CSF, or PBS showed cytolytic values of 19,5, 2, or 1%, respectively. Mice treated with M-CSF + IL-2, IL-2,M-CSF, or PBS, and were not given EL4 showed no anti-EL4 cyto

lytic activity (2, 3, 1, or 0%, respectively). These functional studiessupport our FACS findings (which only measure cell number). Theyalso support the hypothesis that M-CSF + IL-2 together induceT-cells and NK cells that are capable of recognizing and killing EL4

cells in vivo.

DISCUSSION

We chose EL4 for our studies since EL4 cells injected in vivoprovide a well-established model for exploring antitumor responses ofa T-cell cancer. Kinetic studies have shown that EL4 elicits immuno-

logical responsiveness through a variety of effector cells, including

Table 3 Coadministration s.c. of M-CSF and IL-2 enhances various leukocyte subpopulations in the spleen of F.L4-injecled mice as measured by FACSC57Bl/fi mice receiving an i.v. injection of HVSEL4 cells on day 0 were given 14-day pumps containing 20 ftg/day M-CSF + fig/day IL-2. On days 7 and 14. spleens were removed.

Three mice/group were studied wilh the exception of the IL-2-treated group, in which there was only one survivor at day 7. Cells from each group were pooled. Samples of cells weretreated with various tluorochromc-laheled monoclonal antibodies for 1- or 2-eolored immunolluorcscencc as described in "Materials and Methods." and percent positive cells weredetermined by FAC'S. Percentages were multiplied by the total spleen count, and values are reported x \(Th cells/spleen. Since 14-day pumps were inserted on day -3. day 14 was

chosen for study since cytokinc delivery had ceased at this lime.

T cellsNK cell, B cell,

Day7M-CSF+IL-2IL-2M-CSFPBSDay

14M-CSF+IL-2IL-2M-CSFPBS8C5*

marker3638412566237F480*marker15273235"HIS98536NK1+CD.3-marker86ho782463110B220'marker59III!15666685930CD3+NKPG9891341W44227CD4+30386232422014CD8+20284131211410CD3+IL-2R+23282514184CD3+ NK1+33X2712184

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ANTITUMOR EFFECTS WITH M-CSF + H.-2 THERAPY

Fig. 4. Continuous s.c. administration of M-CSF+ IL-2 by pump docs not protect against EL4 tu-

morigcnicity in mice that have been depleted of NKcells i/i vñ'o.Groups of IO C57BI/6 mice that were

given a depleting dose of NK1.1 monoclonal antibody on days -4, 3, and 10 were given pumpscontaining M-CSF + IL-2, M-CSF, IL-2, or PBSon day -3 and injections of IO5 EL4 cells i.V. onday 0. Other groups of mice were depleted of T-cells by adult thymcctomy and injection of anti-CD4 (GK1.5) and anti-CDH (anti-Lv2.2) on days-14 and -7, or were not depleted. Actuarial sur

vival was plotted.

0)

CoCoo.oo!

1.0-5

0.9-

0.8-

0.7-

0.6

0.5

0.4

0.3

0.2-

0.1 -i

lp1•M-CSF+IL

NKdepict•PBS

NK deplc<Y129'»ten\.*p<0.006

compared to PBS/nondeplete__tp<0.03 compared toM-CSF+IL-2/nondepleteH

1 1M-CSF+IL-2/nondeplete

iII

^M-CSF+IL-2/TdepleteliPBS/nondepletePBS/T

deplete

10 20 30 40 50 60 70 80 90 100

Days

cytolytic T-cells, NK cells, LAK cells, and suppressor cells (39). EL4was also selected because we were interested in studying the immu-nohiology of M-CSF + IL-2 coadministration without the complication of a tumor that expresses M-CSF receptor (c-fins) and IL-2receptor. Others have reported that EL4 does not express IL-2 receptor(40), and in our own studies we were unable to detect IL-2 receptor

using FACS and could not activate EL4 tumor cells in vitro withrecombinant M-CSF and IL-2. Although this argues that IL-2 is not

acting directly on EL4 cells, we cannot rule out the possibility thatIL-2 receptor ß-or "y-chains may some how allow these cells torespond to IL-2.

We observed that M-CSF + IL-2 coadministration stimulated sev

eral cell populations with potential antitumor effector cell properties.Our data were consistent with an effect on monocytes and macrophages. Phenotyping studies using FACS showed that macrophagenumbers (F4/80* cells) were increased by coadministration as com

pared to macrophage numbers obtained when the cytokines wereadministered individually. This observation was not surprising, sinceinvestigators reported that c-fins and IL-2 receptor are both present onmonocytes (41). Their data demonstrated that IL-2 enhanced c-finsmRNA and c-fms glycoprotein expression, suggesting that IL-2, byaugmenting expression of c-fins, can lead to prolongation of mono-cyte-mediated tumoricidal activity. Investigators have also shown thatperipheral blood cells from cancer patients given IL-2 treatment haveenhanced expression of mRNA for M-CSF (42). It is therefore possible that IL-2 treatment increases M-CSF production, which in turn

increases macrophage numbers and/or macrophage activity in oursystem. Human monocytes in culture with IL-2 have shown high

activities against various tumors (43). Macrophage effector cellsagainst EL4 have previously been reported in mice (39) and in thesestudies, function was diminished in late tumor-bearing mice, suggest

ing that suppression occurs as the tumor expands. Together, thesefindings suggest that macrophage/monocytes could play a primaryrole in the anti-EL4 response, while the role of other cell types couldbe more indirect, i.e., secondary provision of cytokines such as inter-feron-y or tumor necrosis factor that could further activate monocytes.However, in our model, macrophage stimulation with M-CSF alone

was only partially protective against EL4. This suggests that othercells are operative in the antitumor effect of M-CSF + IL-2.

NK cells were also important in the coadministration of M-CSF +IL-2. Phenotyping studies showed that in either normal or EL4-in-fected hosts, the highest level of NK1.1+CD3~ cells was obtained

with M-CSF + IL-2 treatment rather than IL-2 or M-CSF treatmentalone. A more direct link of NK cells to the anti-EL4 leukemia effectwas established in the in vivo depletion experiments using anti-NKl. 1

injections to eliminate NK cells. This established method of depletion(44) significantly augmented the rates of EL4-related deaths and alsototally prevented a successful M-CSF + IL-2 response. M-CSF wassynergistic with IL-2 in inducing NK cell numbers and/or function.Thus, while IL-2 probably had a direct effect stimulating IL-2 receptor-positive NK cells and their precursors, the M-CSF effect waslikely indirect through interferon. M-CSF is known to stimulate the

production of interferon (45), and investigators have established a roleof monocytes in the IL-2 activation of human NK cells (46). It has

been shown that interferon is a potent stimulator of NK activity intumor models (47). Interferon has been shown to act through NK cellsin other EL4 studies (48). Administration of interferon did enhance invivo anti-EL4 effect in normal C57B1/6 mice and in T-cell-deficientnude mice, but did not in NK-deficient beige mice. It may be impor

tant that other investigators (49) have shown that macrophage precursors enriched from murine bone marrow show strong proliferation inresponse to treatment with M-CSF and IL-2 in vitro, and that IL-2exposure induced NK-like activity in these cells. When IL-2 was

withdrawn from the culture media, cells reverted to properties oftypical macrophages.

Interestingly, our phenotyping studies showed that theCD34NK1.1 *•cells (50) that express both NK marker (NK1.1) and

the T-cell receptor, behaved more like T-cells than NK cells: they wereequally expanded by M-CSF + IL-2 treatment or IL-2 treatment

alone. This might not be so surprising in light of the fact that thesecells have been shown to differentiate into a CD3+CD8* NK1.1~

phenotype. Thus, these cells were apparently generated in response toIL-2 and not augmented by the M-CSF + IL-2 combination. Taken

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ANTITUMOR EFFECTS WITH M-CSF + IL-2 THERAPY

together, our data indicate that NK cells are key effector cells in theM-CSF + IL-2 response.

T-cells were also affected in our system. The relationship betweenT-cells and anti-EL4 protection was established in vivo in experimentswith thymcctomized adult mice that were further T-cell-depleted within vivo injections of anti-CD4 and anti-CD8 monoclonal antibody. (A

group of 60 of these mice had a mean of only 1.4% CD4 expressingT-cells and 1.5% CD8 expressing T-cells and no T-cell function.4) In

these mice given EL4 cells, M-CSF + IL-2 treatment resulted in a

40% day % survival rate, which was significantly lower (P < 0.03)than the survival rate in nonthymectomizcd/non-T-cell-dcplctcd micegiven M-CSF + IL-2. Thus, the removal of T-cells diminished, but didnot eliminate M-CSF + IL-2 antitumor efficacy. Previously, a role forspecific T-cell responses in the EL4 model has been reported (48, 51).In our in vitro studies, the highest levels of anti-EL4 CTL functionwere measured from mice given injections of both M-CSF + IL-2 andnot the individual cytokines. In our phenotyping studies of EL4-injected mice, we examined the number of several independent T-cellpopulations in the spleen, including CD3 ' (T-cell receptor positive)cells, CD4' helper T-cells, CDS* CTL, and CD3+ IL-2 receptor-

expressing (activated) T-cells. All were significantly elevated. However, we achieved the same increases in cell numbers with IL-2 aloneas we did with the M-CSF + IL-2 combination. Identical findings

were obtained in separate experiments in normal mice treated withM-CSF + IL-2. Together, these data indicate that although IL-2 alonemight be chiefly responsible for the increase in T-cell number, theM-CSF + IL-2 combination was the cause of the enhanced anti-EL4CTL function and that T-cells are clearly important in the M-CSF +IL-2 response.

Neutrophil levels were also high in M-CSF- and IL-2-trcated mice.

This was shown in stained blood smears and supported by our phenotyping experiments. However, levels were similarly elevated inIL-2-treated mice. Since our previous studies indicated that G-CSF(37, 52) infusion induced neutrophilia in mice, we administered G-

CSF to mice given EL4 in the same experiment shown in experiment2A. No protection against EL4 was afforded, despite the fact that highlevels of neutrophils were measured in the G-CSF-treated mice on day11. In fact, higher levels of neutrophils were seen in the G-CSF-treatedmice than in the mice given M-CSF + IL-2. Thus, although neutrophillevels are elevated by M-CSF and IL-2 treatment, these cells do not

appear to play an important role in protecting our mice from EL4. Onthe other hand, we cannot rule out an anti-EL4 effect mediated byneutrophils, since M-CSF or the M-CSF + IL-2 combination may be

affecting neutrophil function in a qualitatively different fashion thanthat induced by G-CSF. We do not exactly know why neutrophil levelswere elevated, but it is possible that accessory cytokines such as IL-1,G-CSF, granulocytc macrophage-colony-stimulating factor, and IL-6were elevated by M-CSF + IL-2 treatment. Investigators have shownthat in cancer patients given IL-2 pronounced increases in G-CSF andIL-6 were observed (53).

We found that lower doses of IL-2 cured a percentage of mice giveninjections of EL4. However, at higher IL-2 doses, early toxic deaths

were observed. This finding was not surprising in light of clinicalstudies demonstrating vascular leak syndrome and other toxic effectsrelated to clinical IL-2 treatment (54). We know that IL-2 can inducethe release of other cytokines such as tumor necrosis factor (55), IL-1(56), and intcrferon-y (57), which can be toxic. We do not know whyM-CSF was able to protect mice from the early IL-2-reIated toxicdeaths. One could speculate that M-CSF stimulates the monocyte pooland increases their numbers. Since IL-2 stimulates IL-2 receptor expression on monocytes (58), IL-2 might preferentially bind to thisexpanded monocyte pool. Less IL-2 might be available to bind to

4 D. A. Vallera el al., unpublished.

other targets that stimulate cytokine release and toxicity. Perhaps theaddition of M-CSF to IL-2 changes the cytokine profile. Future stud

ies will be performed to learn more about the role of other cytokinesin mice given M-CSF + IL-2. We do not favor the explanation that theM-CSF protein directly slowed the release of IL-2 from the pump,because phenotypic studies show that M-CSF + IL-2 promote different populations of cells than either M-CSF or IL-2 treatment alone.For example. F4/804 macrophages and NK1.T CD3~ NK cells were

highest with M-CSF + IL-2 treatment. Also, protection against thetumor was obtained when M-CSF and IL-2 were delivered in separate

pumps placed in the same animal (data not shown). Our in vivoM-CSF dose of 20 ftg/day was derived from our previous mouse

studies (59). A dose of 30 /xg/day was toxic.In conclusion, our data support an important role for macrophages,

T-cells. and NK cells in the anti-EL4 response induced by the M-CSF+ IL-2 combination. Macrophages are important since macrophage

numbers were highest when the combination was used. Also it isknown that IL-2 enhances c-fhix expression, and receptors for bothcytokines are found on macrophages. Our data also show that T-ccllfunction is important since T-ccll anti-EL4 CTL function was highestin the M-CSF + IL-2 combination (although the removal of T-cellsdid not entirely ablate the efficacy of the M-CSF + IL-2 combinationagainst EL4). Neutrophils appeared to play a minor role in the anti-tumor response. Although neutrophil levels were elevated, G-CSF was

unable to confer any protection against EL4. Thus, their role is debatable. The studies in this manuscript support the argument thatcombined M-CSF + IL-2 therapy represents a strong immunostimu-

latory regimen, which, based on findings in an aggressive systemictumor model, shows some degree of promise. The beneficial effect ofthis cytokine combination is not restricted to chemically inducedtumors, since reports show that combined M-CSF + IL-2 treatmentcan be used to protect 7-day-old C57B1/6 mice from HSV-1 infection

(60). Perhaps of greater importance is the observation that the additionof M-CSF rcproducibly diminished toxic deaths attributed to IL-2.This may provide a means to enhance IL-2 dosages in future trials. AnM-CSF + IL-2 regimen may be interesting in light of clinical trialswith IL-2 (13) and M-CSF (61) alone.

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1993;53:4273-4280. Cancer Res   Daniel A. Vallera, Patricia A. Taylor, S. Lea Aukerman, et al.   Stimulating Factor and Interleukin-2Coadministration of Recombinant Macrophage-ColonyLeukemia/Lymphoma EL4 by the Continuous Subcutaneous Antitumor Protection from the Murine T-Cell

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